Respiratory viral infections have been significant threats to human health and lives for centuries. Notorious episodes include infections caused by influenza strains, respiratory syncytial virus, and sever acute respiratory syndrome (SARS). These include the global influenza pandemic of 1918, which killed approximately 20-40 million people worldwide. There have been other influenza pandemics in more recent decades as well. A SARS outbreak in 2002 claimed around 800 lives (2).
Respiratory Syncytial Virus
Respiratory syncytial virus (RSV) infection is the major cause of serious pediatric respiratory tract disease. About two-third of infants are infected with RSV during the first year of life and almost 100% have been infected by age 2. There is currently no specific and effective therapeutic available to treat RSV infection.
Respiratory syncytial virus (RSV) is an enveloped, non-segmented single-strand negative RNA virus (NNR) belonging to the family Paramyxoviradae, in the order mononegaviruses (14, 29). The paramyxoviruses share the following features. 1) They have a single stranded RNA genome that is tightly wrapped with the viral nucleocapsid protein (N)29, 30. 2) Sub-genomic mRNAs are transcribed from the negative genome by RdRP. 3) Virus replication takes place in the cytoplasm of host cells. The details of RSV life cycle from infection to release of progeny virions are well studied (15).
The RSV genome is a negative strand 15.5 kb long, containing the genes 3′-NS1, NS2, N, P, M, SH, G, F, M2, L-5′ (see FIG. 1). Of these seven gene products are common to other paramyxoviruses 3, namely, N, P, SH, G, F, and L. Several viral or host factors are involved in the regulation of RNA transcription and replication (20). There are in addition viral cis-acting signals that play regulatory roles in transcription of mRNAs and RNA replication (3).
The incubation period for RSV infection is about 4 to 5 days; it first affects the nasopharynx, then in a few days it reaches the bronchi and bronchioles, with infection confined to the superficial layer of the respiratory epithelium.
Influenza A
Beginning in 1997, a new strain of avian influenza A, H5N1, has appeared. Although confined mostly to fowl, both wild populations and domesticated birds, the virus infects humans apparently only be direct contact with infected birds. In humans infection causes serious disease, leading to severe respiratory illness and death in human beings (3-12). Numerous cases and outbreaks have occurred in various nations of southeast Asia. In view of the ability of the avian virus to infect humans, there is increased risk of mutation to a contagious human variant, risking the emergence of a new influenza pandemic with efficient and sustained human-to-human transmission, and significant mortality.
Since avian flu H5N1 is a newly emerging infectious agent associated with pneumonia and its pathology and mechanism is not very clear, there is no specific and effective treatment for H5N1 avian flu in the human disease cases yet. Currently influenza infections are treated with antivirals, such as the two drugs (in the neuraminidase inhibitors class), oseltamivir (commercially known as Tamiflu) and zanamivir (commercially known as Relenza), or the older M2 inhibitors amantadine and rimantadine.
H5N1 is a subtype of influenza virus type A. As such it is an enveloped, fragmented, negative-single stranded RNA virus, belonging to the family Orthomyxoviridae. During the life cycle of the influenza A virus (including H5N1), the viral genome RNA (vRNA) serves as a template for complementary RNA (cRNA) production, which also serves as the template for messenger RNA (mRNA) production. Each of these three forms of RNA molecules arising during viral replication can all be targeted for siRNA-mediated degradation, using either sense or antisense siRNAs. The influenza A genome, consisting of 8 separate RNA segments containing at least 10 open reading frames (ORFs), serves as template for both viral genome replication and subgenomic or gene-directed mRNA synthesis. FIG. 2 shows a diagram representing the structure of an influenza A virion. Polymerases PB2, PB1 (polymerase basic protein 1 and 2) and PA (polymerase acidic protein) were coded by RNA1, RNA2 and RNA3 respectively. Four viral structural proteins H (hemagglutinin), N (neuraminidase), M1 and M2 (matrix proteins 1 and 2) are respectively coded by RNA segments 4, 6 and 7, while RNA5 codes for NP (nucleocapsid protein) and RNA8 codes for NS1 and NS2 (nonstructural proteins 1 and 2).
RNA Interference
RNA interference (RNAi) is a sequence-specific RNA degradation process that provides a relatively easy and direct way to knockdown, or silence, theoretically any gene (17, 18, 19). In naturally occurring RNA interference, a double stranded RNA is cleaved by an RNase III/helicase protein, Dicer, into small interfering RNA (siRNA) molecules, a dsRNA of 19-23 nucleotides (nt) with 2-nt overhangs at the 3′ ends. These siRNAs are incorporated into a multicomponent-ribonuclease called RNA-induced-silencing-complex (RISC). One strand of siRNA remains associated with RISC, and guides the complex towards a cognate RNA that has sequence complementary to the guider ss-siRNA in RISC. This siRNA-directed endonuclease digests the RNA, thereby inactivating it. Recent studies have revealed that the use of chemically synthesized 21-25-nt siRNAs exhibit RNAi effects in mammalian cells 20, and the thermodynamic stability of siRNA hybridization (at terminals or in the middle) plays a central role in determining the molecule's function (21, 22). These and other characteristics of RISC, siRNA molecules and RNAi have been described (23-28).
Application of RNAi in mammalian cells in laboratory or potentially, in therapeutic applications, uses either chemically synthesized siRNAs or endogenously expressed molecules (2, 21). The endogenous siRNA is first expressed as a small hairpin RNAs (shRNAs) by an expression vector (plasmid or virus vector), and then processed by Dicer into siRNAs. It is thought that siRNAs hold great promise to be therapeutics for human diseases especially that caused by viral infections (19, 20, 27-30).
Importantly, it is presently not possible to predict with any degree of confidence which of many possible candidate siRNA sequences potentially targeting a viral genome sequence (e.g., oligonucleotides of about 16-30 base pairs) will in fact exhibit effective siRNA activity. Instead, individual specific candidate siRNA polynucleotide or oligonucleotide sequences must be generated and tested to determine whether the intended interference with expression of a targeted gene has occurred. Accordingly, no routine method exists in the art for designing a siRNA polynucleotide that is, with certainty, capable of specifically altering the expression of a given mRNA.
There remains a significant need for compositions and methods that inhibit expression of viral pathogen genes and their cognate protein products. In particular there is an urgent need for compositions and methods to inhibit expression of pathogenic respiratory viral genes in virus-infected-cells, and for treating a respiratory viral infection in a subject. There further is a need for compositions and methods addressing infection by RSV and avian influenza A, especially the H5N1 strain. There additionally is a need for compositions and methods for treatment that are highly effective, and do not rely on use or modification of known antiviral agents. The present invention addresses these and related needs.